Recently, an ideal treatment method for cancer, with minimal side effects, which does not affect normal cells but can selectively injure only cancer cells, is needed. For example, a gene therapy method that can enhance the selectivity of cancer cells in various levels, such as the cell selectivity of a gene to be inserted into cancer cells, the activity of an expression promoter, the method of introduction and infection of a virus vector, or the like, is regarded as a promising treatment method. However, a common problem of the existing methods is that the therapeutic gene cannot be introduced into all cancer cells.
On the other hand, concerning the immunocytic therapy of cancer, slight expression of a tissue-specific differentiation antigen has also been observed in normal tissue. Unfortunately, the expression in normal cells presents a problem. A cancer antigen based on a mutation has the disadvantage that this mutation is directed to the specific cancer, and it is therefore not appropriate to generalize it as an immunocytic therapy for cancer as a molecular target.
Recently, a clinical study was conducted in the United States and in Great Britain (Gene Ther. 7, 859-866, 2000; Gene Ther. 7, 867-874, 2000), respectively demonstrating gene therapy to treat malignant brain tumors using a replication-competent herpes simplex virus (HSV) vector, which selectively injures only the continuously growing cells by infection and replication. The replication-competent HSV vector is a vector wherein a ribonucleotide reductase (RR) or thymidine kinase (TK), essential to viral replication, is deleted. These enzymes are expressed only at the time of proliferation in normal cells, but are constitutively expressed in tumor cells. Thus, when the HSV vector infects actively proliferating cells, whether it is a normal cell or a tumor cell, the vector is replicated using cell-derived RR or TK, and shows cytolysis activity. Anti-tumor effects of a replication-competent HSV vector, with respect to prostate cancer and pancreatic cancer, have also been reported in animal experiments. (J. Surg. Oncol. 72, 136-141, 1999). However, these also do not have cell selectivity and the safety level is not high. Therefore, it could be used for treating tumor cells in the human brain, which has a blood brain barrier and where the vector does not diffuse into circulating blood. The problem is that this therapy is not appropriate for the treatment of organs other than the brain.
According to the discussion above, it is thought that if it were possible to control the injury activity of the HSV vector to target specific cells, it could be a more effective and safe treatment method. Miyatake and Martuza et al., of the United States, have reported a replication-competent HSV vector selective for liver tumor using an albumin promoter (J. Virol. 71, 5124-5132, 1997). However, when such a vector is used, the expression of the albumin gene decreases in liver cell carcinoma and also injures normal regenerated liver cells. Therefore, this treatment is considered to be inappropriate for clinical application in humans. Furthermore, Martuza and Miyatake have reported the possibility of this clinical application to mesothelioma in U.S. Pat. No. 5,728,379 (“Tumor- or cell-specific herpes simplex virus replication”), patented in March 1998. Notably, there is no description of the possible clinical application to human sarcoma in general, for example, leiomyosarcoma, osteosarcoma, and the like.
Gene analysis of the cause of disease and the pathology of sarcoma found a mutation of p53 and Rb and the existence of a fusion gene in some of the tumors, but the results are not yet at the stage to be applied widely for treatment. In an animal experiment using nude mice, Milas et al., have introduced the p53 gene into leiomyosarcoma cells, using an adenovirus vector that does not have the ability to replicate, and have reported delayed tumor proliferation (Cancer Gene Ther. 7, 422-429, 2000). Furthermore, a method for introducing and expressing thymidine kinase, a suicide gene, into osteosarcoma using a promoter of osteocalcin gene has been reported (Cancer Gene Ther. 5, 274-280, 1988). However, this method uses a virus vector where the ability to replicate is removed, and the efficiency of introducing the gene is low. Thus, this method cannot be applied to sarcoma other than osteosarcoma.
Especially, in the report of Milas et al., an example using the human smooth muscle cell line SK-LMS-1 is described. However, the number of viral particles used is more than 100 to 1000 times compared to the number of particles of virus vector used in the present invention. Thus, the effect is inferior compared to the example of the present invention. Therefore, the results of Milas, et al. are not preferable since the number of viral particles to be injected into the body should be minimal to reduce side effects.
Moreover, as for a therapy for suppressing vascularization of cancer, Folkman et al. have reported a dramatic anti-tumor effect of peptidergic inhibiting factors such as angiostatin, endostatin or the like, in mice experiments (Cell 79, 315-328, 1994; Cell 88, 277-285, 1997). Nakamura et al. have also reported the action to suppress vascularization of NH4, which is a fragment of a liver cell growth factor molecule (Biochem. Biophys. Res. Commun. 279, 846-852, 2000). However, these methods are problematic and have problems such as, for example, a large required number of peptides; low endostatin reproducibility; unknown mechanism of action; and unconfirmed efficacy in humans, or the like. A vascularization inhibitor, now being clinically tested, does not have cell selectivity and has low inhibition efficiency. The peptide inhibiting integrin from acting on the surface of endothelial cells, which Cheresh et al. of the United States has reported, similarly does not have cell selectivity and the inhibition efficiency is low (J. Clin. Invest. 103, 1227-1230, 1999). All of these studies are directed to treatments where the target is a vascular endothelial cell. However, a therapeutic agent having cell selectivity targeting proliferating vascular smooth muscle cells in tumor vessels remains unknown. Actually, the antagonist of a platelet-derived growth factor receptor, which promotes the proliferation and the migration of smooth muscle cells, has been reported to have a strong tumor neovascular suppressing action (Cancer Res. 60, 4152-4160, 2000). The significance of attacking vascular smooth muscle cells to suppress tumor vascularization is being evaluated, but since this method is not cell specific, disadvantages or problems are anticipated.
On the other hand, the calponin gene, which is said to be a differentiated marker of smooth muscle, was found to be expressed in tumor cells of human-derived sarcoma (Int. J. Cancer 79, 245-250, 1998; Sarcoma 3, 107-113, 1999; Intern. J. Cancer 82, 678-686, 1999). Many reports have been subsequently made, indicating that the calponin gene is expressed abnormally in about 20 kinds of human malignant tumors derived from mesenchymal cells, such as gastrointestinal stromal tumor (GIST), salivary gland sarcoma, fibrosarcoma, malignant neurilemmoma and the like, in addition to sarcoma in the bone and soft parts. The calponin gene mentioned above (h1 or basic) has been shown by X-ray crystalography and in vitro and in vivo mechanism analyses to suppress the sliding movement of actin/myosin by binding to the C-terminus of the actin molecule (Biochem. Biophys. Res. Commun. 279, 150-157, 2000; J. Physiol. 529, 811-824, 2000). The calponin gene is considered to be selectively expressed in smooth muscle cells of an adult and is a differentiated marker in blood vessels (Physiol. Rev. 75, 487-517, 1995).